Overall aim of this grant is to elucidate the causal relationship between endothelial dysfunction, oxidative stress, and the inflammatory cytokine, tumor necrosis factor alpha (TNFot). Accordingly, this proposal will test the hypothesis that inflammation produces endothelial dysfunction in Type II diabetes, which is a leading risk factor for coronary heart disease. There are three aims, and associated with each of the aims are three or four hypotheses. The first aim will determine if TNFa plays a critical role in diabetic endothelial dysfunction. We propose that: 1). In dbTNF7dbTNF" mice endothelial dilation will be greater than that in diabetic (db/db) mice, and comparable to Db/db controls. Decrements in endothelial function in db/db mice will progressively increase from 6 to 12 to 18 weeks. 2). Administration of neutralizing antibodies to TNFa will ameliorate endothelial dysfunction in db/db mice of varying ages, 6, 12 and 18 weeks. The effect will be most prominent in the older animals. 3). Expression of TNFa and TNFa receptors (TNFRs: TNFR1 and TNFR2) are elevated in coronary arterioles, even at the age of 6 weeks before the development of diabetes, in db/db mice compared to Db/db mice. We propose that the expression of TNFa and its receptors increase progressively with age, and with the development of diabetes. The second aim will determine if TNFa produces endothelial dysfunction by stimulation of the production of reactive oxygen species (ROS) in db/db mice through activation of NADPH oxidase. We propose to test anti-TNFa attenuate ROS production in diabetes in db/db mice. The third aim will determine if AGE/RAGE and TNFa signaling interact to amplify the oxidative stress and induce endothelial dysfunction in diabetic mice. We propose to test if: 1). In db VdbTNF" mice, RAGE expression and AGE formation are less than in db/db mice. 2). Activation of the transcription factor NFicB is less in dbTNF"/dbTNF' or db/db + sRAGE mice compared to db/db mice. 3). In db/db mice of varying ages: 6, 12 and 18 weeks, sRAGE restores endothelial dependent dilation toward that of Db/db controls by reducing TNFa expression and ROS production. 4). Activation of RAGE compromise endothelial dilation to a greater extent in db/db than in Db/db or dbTNF"/dbTNF" mice. 5). In db/db mice, sRAGE reduce ROS production and TNFa expression compared to that in db/db mice, and this effect will be mediated through decreased expression of NAD(P)H oxidases. We utilize a combination of approaches involving diameter measurements of isolated coronary arterioles from mouse heart, the oxidative fluorescent dye dihydroethidium (DHE-fluorescence imaging) and electron paramagnetic resonance (EPR) to evaluate O2" production, electrochemical detection of authentic NO, real time PCR of RNA transcripts, and Western Blotting to evaluate expression of key proteins in Db/db control, dbTNF"/dbTNF", db/db + sRAGE and db/db mice. We believe that this study will provide a solution to the specific problem and new approaches for the longer term, it might represent exciting new targets for the development of drugs for cardiovascular disorders in ischemic heart disease, obesity, diabetes and hypertension.